Mechanism of the T286A-Mutant αCaMKII Interactions with Ca²⁺/Calmodulin and ATP

Abstract

The role of adenosine 5'-triphosphate (ATP) in the activation mechanism of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) was investigated using the T286A non-autophosphorylatable mutant of alphaCaMKII. Characterization of the T286A-alphaCaMKII mutant revealed k(cat) = 0.06 +/- 0.02 s(-1) for the T286A mutant, a 6 (+/- 2)-fold lower value compared to wild-type alphaCaMKII with 100 microM smooth muscle myosin light chain (MLC) as substrate. MLC phosphorylation by the T286A mutant and wild-type alphaCaMKII was cooperative, with Hill coefficients 2.3 +/- 0.1 and 2.4 +/- 0.3, respectively. K(m) values for MLC were 96 +/- 28 microM with T286A-alphaCaMKII and 49 +/- 29 microM for wild-type alphaCaMKII. Thus, while the activity of alphaCaMKII was sensitive to mutation of the Thr(286) residue to Ala, the mechanisms of the wild-type and T286A mutant enzyme appeared similar. K(d) for Ca(2+)/calmodulin was 2-fold reduced to 40 nM compared to that of wild-type alphaCaMKII (75 nM). ATP induced a 9-fold stabilization of Ca(2+)/calmodulin binding to the T286A mutant enzyme. Fluorescence stopped-flow kinetic experiments revealed that two Ca(2+)/calmodulin-enzyme complexes were formed, the first, unaffected by ATP, with association and dissociation rate constants of 2 x 10(7) M(-1) s(-1) and 5 s(-1), respectively, containing calmodulin in extended conformation. The second complex, in which calmodulin adopted a compact conformation, was formed with association rate constant 3 x 10(6) M(-1) s(-1) and dissociation at 0.15 s(-1) in the absence and 0.015 s(-1) in the presence of ATP. These data show that ATP is involved in the activation mechanism by forming two classes of Ca(2+)/calmodulin.alphaCaMKII.ATP complex. It is likely that only one of the complexes is on the activation pathway.

An allosteric model of calmodulin explains differential activation of PP2B and CaMKII

“…This allows for some calmodulin activity even at low calcium concentrations. Furthermore, together with the facts that calmodulin affinity for PP2B (31) is higher than for CaMKII (32) and that CaMKII concentration in the PSD (40) is higher than PP2B concentration (41), it can explain why PP2B is preferentially activated at lower calcium concentrations. Models of this type do not allow for the possibility that the different binding sites might have different affinities for calcium.…”

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

“…The underlying mechanism is unclear, and the kinase domain structure of CaMKII has been solved so far only in absence of nucleotides (55). However, nucleotide can enhance Ca 2ϩ /CaM binding independent of T286 autophosphorylation (57), possibly by facilitating displacement of the regulatory helix, which would make the T-site more accessible. Additionally, nucleotide may induce a conformation in or around the T-site that is more favorable for external interaction.…”

Dual Mechanism of a Natural CaMKII Inhibitor

“…2-Chloro-(-amino-Lys 75 )-[6-(4-N,N-diethylaminophenyl)-1,3,5-triazin-4-yl] calmodulin (TA-CaM) was kindly provided by Dr. Katalin Torok Tzortzopoulos and Torok, 2004). Green fluorescent protein (GFP)-CaMKII␣ wild-type and mutants were expressed in Cos-7 cells, and extracts were prepared in 50 mM piperazine-N,NЈ-bis(2-ethanesulfonic acid) (PIPES), pH 7.2, 10% glycerol, 1 mM EGTA, 1 mM dithiothreitol, and protease inhibitors (Roche Diagnostics, Indianapolis, IN) .…”

“…TA-CaM dissociation was assessed by increased TA-CaM (30 nM) fluorescence after dissociation from CaMKII or its CBD (150 nM) during a chase with unlabeled CaM (60 M) Tzortzopoulos and Torok, 2004). Buffer contained 50 mM HEPES, pH 7.4, 150 mM KCl, 2 mM MgCl 2 , 2 mM Mg-ADP, 2 mM CaCl 2 , and 0.1 mg/ml BSA.…”

“…We hypothesized that CaM-KIIN and CN21a should have a similar effect, if CaM trapping is indeed mediated by displacement of the CaMKII autoregulatory region from the T-site. To follow CaM dissociation, we used TA-CaM, which shows increased fluorescence after dissociation from CaMKII or a peptide derived from the CaMKII calmodulin binding domain (CBD) Tzortzopoulos and Torok, 2004). Dissociation of TACaM was initiated by a chase with an excess of unlabeled CaM, and the change in fluorescence was followed over time ( Figure 4).…”